Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

• Published in: Canadian journal of microbiology Vol. 58; no. 9; pp. 1035 - 1046
• Main Authors: Sørensen, Annette, Ahring, Birgitte K, Lübeck, Mette, Ubhayasekera, Wimal, Bruno, Kenneth S, Culley, David E, Lübeck, Peter S
• Format: Journal Article
• Language: English
• Published: Ottawa, ON NRC Research Press 01.09.2012; National Research Council of Canada
• Subjects: Amino Acid Sequence; amino acids; Aspergillus; Aspergillus - enzymology; Aspergillus - genetics; Aspergillus aculeatus; Aspergillus niger; Aspergillus saccharolyticus; beta-glucosidase; beta-Glucosidase - chemistry; beta-Glucosidase - genetics; beta-Glucosidase - metabolism; Biological and medical sciences; biomass hydrolysis; caractérisation d’enzyme; cellobiose; Cellobiose - metabolism; Cellulose - analogs & derivatives; Cellulose - metabolism; culture media; Dextrins - metabolism; Enzymatic analysis; enzyme characterization; enzyme kinetics; Fundamental and applied biological sciences. Psychology; fungi; Fysik; genes; Genetic aspects; Glycosidases; glycosides; Half-Life; Hydrogen-Ion Concentration; hydrolyse de la biomasse; Hydrolysis; Hypocrea jecorina; Identification and classification; ion exchange chromatography; Kinetics; mass spectrometry; Microbiology; Miscellaneous; Models, Molecular; Molecular Sequence Data; Mycology; Natural Sciences; Naturvetenskap; Observations; Physical Sciences; polyacrylamide gel electrophoresis; polymerase chain reaction; polypeptides; Properties; Protein Structure, Tertiary; Sequence Alignment; Temperature; thermal stability; thermostability; thermostabilité; Trichoderma - genetics; Trichoderma reesei; β-glucosidase; Denmark; United States; 4-α-Glucanotransferase; Enzyme; Transferases; Glycosyltransferases; Biomass; Fungi; Glycosylases; Hydrolysis; Aspergillus; β-Glucosidase; Glycosidases; Hydrolases; Hexosyltransferases; Fungi Imperfecti
• ISSN: 1480-3275; 0008-4166; 1480-3275
• DOI: 10.1139/w2012-076

The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A. saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS–PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A. saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger, respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50 °C, and at 60 °C it had a half-life of approximately 6 h.